Based on a system for rapid measurement of multi-wavelength transmission spectra of bacteria, multi-wavelength transmission spectra of Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli in different concentrations of 220 to 900nm were obtained. The calibration curves of three kinds of bacteria based on the different wavelengths and the whole spectral band were established and the detection limits of Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli were calculated. The results were compared with those measured by ultraviolet-visible spectrophotometer. The results showed that the linear correlation coefficient between the experimental system and the ultraviolet-visible spectrophotometer was above 0.999 8. In addition, single measurement time of the average transmission spectrum measured by the system was 15 s, which was 30 times of the signal acquisition. By analysis based on the experimental system, the detection limits of three kinds of bacteria at different wavelengths of 220, 300, 350, 400, 500, 550 nm and full spectrum were all better than those of ultraviolet-visible spectrophotometer. And the detection limit calculated by the full spectrum of the multi-wavelength transmission spectra was the lowest. The detection limit of Klebsiella pneumoniae, Staphylococcus aureus and Escherichia coli were 1.60×104, 1.06×104 and 1.16×104 cells·mL-1. The results provide the basic data for the further development of the rapid and quantitative detection technology of the multi-wavelength transmission spectra of bacteria in water.